MTF1 Is Essential for the Expression of MT1B, MT1F, MT1G, and MT1H Induced by PHMG, but Not CMIT, in the Human Pulmonary Alveolar Epithelial Cells

toxics

Article MTF1 Is Essential for the Expression of MT1B, MT1F, MT1G, and MT1H Induced by PHMG, but Not CMIT, in the Human Pulmonary Alveolar Epithelial Cells

Sang-Hoon Jeong 1,†, Cherry Kim 2,†, Jaeyoung Kim 1, Yoon-Jeong Nam 1, Hong Lee 1 , Ariunaa Togloom 1, Ja-Young Kang 1, Jin-Young Choi 1, Hyejin Lee 1, Myeong-Ok Song 1, Eun-Kee Park 3, Yong-Wook Baek 4, Ju-Han Lee 5,* and Ki-Yeol Lee 2,*

1 Medical Science Research Center, Ansan Hospital, Korea University College of Medicine, Ansan-si 15355, Korea; [email protected] (S.-H.J.); [email protected] (J.K.); [email protected] (Y.-J.N.); [email protected] (H.L.); [email protected] (A.T.); [email protected] (J.-Y.K.); [email protected] (J.-Y.C.); [email protected] (H.L.); [email protected] (M.-O.S.) 2 Department of Radiology, Ansan Hospital, Korea University College of Medicine, Ansan-si 15355, Korea; [email protected] 3 Department of Medical Humanities and Social Medicine, College of Medicine, Kosin University, Busan 49267, Korea; [email protected] 4 Humidifier Disinfectant Health Center, Environmental Health Research Department, National Institute of Environmental Research, Incheon 22689, Korea; [email protected] 5 Department of Pathology, Ansan Hospital, Korea University College of Medicine, Ansan-si 15355, Korea * Correspondence: [email protected] (J.-H.L.); [email protected] (K.-Y.L.) † These authors contributed equally to this work. Citation: Jeong, S.-H.; Kim, C.; Kim, J.; Nam, Y.-J.; Lee, H.; Togloom, A.; Kang, J.-Y.; Choi, J.-Y.; Abstract: The inhalation of humidifier disinfectants (HDs) is linked to HD-associated lung injury Lee, H.; Song, M.-O.; et al. MTF1 Is (HDLI). Polyhexamethylene guanidine (PHMG) is significantly involved in HDLI, but the correlation Essential for the Expression of MT1B, between chloromethylisothiazolinone (CMIT) and HDLI remains ambiguous. Additionally, the MT1F, MT1G, and MT1H Induced by differences in the molecular responses to PHMG and CMIT are poorly understood. In this study, PHMG, but Not CMIT, in the Human RNA sequencing (RNA-seq) data showed that the expression levels of metallothionein-1 (MT1) Pulmonary Alveolar Epithelial Cells. isoforms, including MT1B, MT1E, MT1F, MT1G, MT1H, MT1M, and MT1X, were increased in human Toxics 2021, 9, 203. https://doi.org/ pulmonary alveolar epithelial cells (HPAEpiCs) that were treated with PHMG but not in those 10.3390/toxics9090203 treated with CMIT. Moreover, upregulation of MT1B, MT1F, MT1G, and MT1H was observed only in PHMG-treated HPAEpiCs. The protein expression level of metal regulatory transcription factor 1 Academic Editor: Jeroen Vanoirbeek (MTF1), which binds to the promoters of MT1 isoforms, was increased in PHMG-treated HPAEpiCs but not in CMIT-treated HPAEpiCs. However, the expression of early growth response 1 (EGR1) Received: 16 June 2021 Accepted: 27 August 2021 and nuclear receptor superfamily 3, group C, member 1 (NR3C1), other transcriptional regulators Published: 29 August 2021 involved in MT1 isomers, were increased regardless of treatment with PHMG or CMIT. These results suggest that MTF1 is an essential transcription factor for the induction of MT1B, MT1F, MT1G, and

Publisher’s Note: MDPI stays neutral MT1H by PHMG but not by CMIT. with regard to jurisdictional claims in published maps and institutional afﬁl- Keywords: polyhexamethylene guanidine; chloromethylisothiazolinone; metallothionein 1; metal iations. regulatory transcription factor 1; human pulmonary alveolar epithelial cells

Copyright: © 2021 by the authors. 1. Introduction Licensee MDPI, Basel, Switzerland. Fatal lung injury is one of the most harmful effects caused by the inhalation of hu- This article is an open access article midiﬁer disinfectants (HDs), which are compounds used to prevent the growth of microor- distributed under the terms and ganisms such as bacteria and fungi, inside the humidiﬁers. Polyhexamethylene guanidine conditions of the Creative Commons (PHMG), oligo(2-(2-ethoxy)ethoxyethyl) guanidinium chloride, chloromethylisothiazoli- Attribution (CC BY) license (https:// none (CMIT), and methylisothiazolinone are the most common HDs [1,2]. Although creativecommons.org/licenses/by/ exposure to PHMG has clearly resulted in lung injuries, such as interstitial pneumonitis 4.0/).

Toxics 2021, 9, 203. https://doi.org/10.3390/toxics9090203 https://www.mdpi.com/journal/toxics Toxics 2021, 9, 203 2 of 10

and fibrosis, CMIT is not considered to be correlated with lung injuries in humans [1,2]. Although a few clinical and animal studies have reported that lung damage after inhalation of CMIT is lower than that observed after inhalation of PHMG [3,4], the differences in the molecular mechanisms between PHMG and CMIT have not yet been fully elucidated. In vitro studies have revealed the significant involvement of PHMG in apoptotic cell death. The increased levels of reactive oxygen species after PHMG inhalation exert cytotoxic effects, such as membrane and DNA damage [5–8]. Furthermore, PHMG has been shown to cause endoplasmic reticulum stress via mitochondrial dysfunction [9]. Exposure to PHMG upregulates the expression of genes involved in various biological processes including apoptosis, autophagy, fibrosis, and cell cycle and downregulates the expression of antioxidant-related genes [10]. PHMG augments the epithelial-mesenchymal transition- related microRNA expression, which may play a role in lung fibrosis [11]. Apoptosis and inflammation have been reported after CMIT exposure; however, CMIT has a weaker association with cellular toxicity than PHMG [12]. Moreover, no study has compared the cytotoxic mechanisms of PHMG with those of CMIT. Metallothioneins (MTs) are small cysteine-rich proteins that bind to intracellular metal ions, such as Zn2+, Cu2+, and Cd2+, to maintain cellular metal homeostasis. The genes encoding these proteins are expressed in response to oxidative stress and metal-induced toxicity [13]. Animal studies have demonstrated that MTs play protective roles in the lungs against multiple oxidative stress-causing factors, such as carmustine [14], ozone [15], paraquat [16], bacterial endotoxins [17], and hypoxia [18]. In contrast, the expression of metallothionein-1 (MT1) is upregulated in different tumors, such as breast cancer, ovarian cancer, urinary bladder cancer, melanoma, and lung cancer [19]. In particular, MT expression level is increased in different types of histological lung cancers, such as squamous cell lung carcinoma and adenocarcinoma [20]. In this study, we analyzed gene expression levels in human pulmonary alveolar epithelial cells (HPAEpiCs) after treatment with PHMG or CMIT. We found that, compared to CMIT, PHMG exposure upregulated the MT1 isomers, including MT1B, MT1F, MT1G, and MT1H, in HPAEpiCs. We also identified that metal regulatory transcription factor 1 (MTF1) is an essential transcription factor for the upregulation of MT1B, MT1F, MT1G, and MT1H in HPAEpiCs treated with PHMG, but not in those treated with CMIT.

2. Materials and Methods 2.1. Chemicals PHMG (CAS No. 89697-78-9) and CMIT (CAS No. 26172-55-4) were purchased from BOC Sciences (Shirley, NY, USA) and Key Organics-Bionet Research (Camelford, UK), respectively.

2.2. Cell Culture HPAEpiCs were purchased from ScienCell (Carlsbad, CA, USA) and cultured in an alveolar epithelial cell medium (ScienCell) supplemented with 1% penicillin/streptomycin ◦ and 10% fetal bovine serum in a 5% carbon dioxide (CO2) incubator at 37 C.

2.3. Cell Viability Assay Cell viability was measured using the Cell Counting Kit-8 (CCK-8; Dojindo, Ku- mamoto, Japan) in accordance with the manufacturer’s instructions. Brieﬂy, HPAEpiCs (1 × 104 cells) were seeded in a 96-well culture plate (SPL Life Science, Gyeonggi, Korea). After treatment with PHMG and CMIT for 12, 24, and 48 h, the HPAEpiCs were incubated with CCK-8 reagent for 2 h. The absorbance of the wells was measured at 450 nm using a microplate reader (SpectraMax M2e; Bucher Biotec, Basel, Switzerland).

2.4. Reverse Transcription-Polymerase Chain Reaction (RT-PCR) Total RNA was extracted from HPAEpiCs using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Complementary DNA was synthesized using the amﬁRivert cDNA Synthesis Toxics 2021, 9, 203 3 of 10

Platinum Master Mix (GenDEPOT, Baker, TX, USA) in accordance with the manufacturer’s instructions. Ampliﬁcation was performed on a thermal cycler (Eppendorf, Hamburg, Germany), and the DNA bands were visualized using a Gel Doc EZ imager (Bio-Rad Laboratories Inc., Hercules, CA, USA). MT1 primers used in this study were designed according to the process described by Mao et al. [21]: MT1B: sense 50-GATCCCAACTGCTCCTGCACCACA-30, antisense 50-AAGAATGTA- GCAAACCGGTCAGGGTAGTT-30; MT1E: sense 50-CACTGGTGTGAGCTCCAGCATCC-30, antisense 50-AATGCAGCAA- ATGGCTCAGTGTTGTATTT-30; MT1F: sense 50-GAATGTAGCAAATGGGTCAAGGTG-30, antisense 50-TCTCCTGCA- CCTGCGCTGGT-30; MT1G: sense 50-CAACTGCTCCTGTGCCGCTGG-30, antisense 50-TGTAGCAAAGGG- GTCAAGATTGTAGC-30; MT1H: sense 50-ATCTGCAAAGGGGCGTCAG-30, antisense 50-GAATGTAGCAAAT- GAGTCGGAGTT-30; MT1M: sense 50-TCCGGGTGGGCCTAGCAGTCG-30, antisense 50-AATGCAGCAAA- TGGCTCAGTATCGTATTG-30; MT1X: sense 50-GACCCCAACTGCTCCTGCTCG-30, antisense 50-GATGTAGCAAAC- GGGTCAGGGTTGTAC-30.

2.5. Western Blotting HPAEpiCs were lysed in RIPA lysis buffer (ATTO, Tokyo, Japan). The protein extracts were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and trans- ferred onto a polyvinylidene ﬂuoride membrane (ATTO). After non-speciﬁc binding to the membrane was blocked by incubation in 5% skim milk for 1 h, the membrane was incubated overnight with anti-MTF1 (1:1000; Abcam, Cambridge, UK), anti-EGR1 (1:1000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), anti-nuclear receptor superfamily 3, group C, member 1 (NR3C1; 1:1000; Santa Cruz), anti-Lamin B1 (1:1000; Abcam), and anti-α-tubulin (1:1000; Santa Cruz) antibodies. An appropriate horseradish peroxidase- conjugated secondary antibody (1:2000; Cell Signaling Technology, Danvers, MA, USA) was used to bind to the primary antibodies, and the protein bands were imaged using the ChemiDoc Touch Imaging System (Bio-Rad Laboratories).

2.6. Library Preparation and Sequencing The quality of extracted RNA was assessed using an Agilent 2100 Bioanalyzer and RNA 6000 Nano Chip (Agilent Technologies, Amstelveen, The Netherlands), and RNA quantification was performed using an ND-2000 spectrophotometer (Thermo Fisher Sci- entific, Inc., Wilmington, DE, USA). For control and test RNAs, library construction was performed using the QuantSeq 30 mRNA-Seq Library Prep Kit (Lexogen, Inc., Vienna, Austria) in accordance with the manufacturer’s instructions. Briefly, 500 ng of the total RNA was obtained and an oligo-dT primer containing an Illumina-compatible sequence at its 50-end was hybridized to the RNA to perform reverse transcription. After degradation of the RNA template, second-strand synthesis was initiated using a random primer containing an Illumina-compatible linker sequence at its 50-end. The double-stranded library was purified using magnetic beads to remove all the reaction components. The library was then amplified to add the complete adapter sequences required for cluster generation. The finished library was purified from other PCR components. High-throughput sequencing was performed as single-end 75 sequencing using NextSeq 500 (Illumina, San Diego, CA, USA).

2.7. Data Analyses QuantSeq 3 mRNA-Seq reads were aligned using Bowtie2 [22]. Brieﬂy, Bowtie2 indices were generated from either the genome assembly sequences or representative transcript sequences to align the reads to the genome and transcriptome, respectively. Toxics 2021, 9, x FOR PEER REVIEW 4 of 11

sequencing was performed as single-end 75 sequencing using NextSeq 500 (Illumina, San Diego, CA, USA).

2.7. Data Analyses

Toxics 2021, 9, 203 QuantSeq 3 mRNA-Seq reads were aligned using Bowtie2 [22]. Briefly, Bowtie2 indi-4 of 10 ces were generated from either the genome assembly sequences or representative transcript sequences to align the reads to the genome and transcriptome, respectively. The alignment file was used to assemble the transcripts, estimate their abundance, and detect the Thedifferential alignment expression file was of used genes. to assemble The differentially the transcripts, expressed estimate genes their were abundance, determined and baseddetect on counts the differential from unique expression and multiple of genes. alignments The differentially using BEDtool expresseds coverage genes [23] were. The de- readtermined count data based were on then counts processed from unique based andon the multiple quantile alignments normalization using method BEDtools using cover- EdgeRage within [23]. The R (Team read count 2013) dataand Bioconductor were then processed [24]. Genes based were on theclassified quantile using normalization the Da- tabasemethod for Annotation, using EdgeR Visualization within R (Team, and 2013) Integrated and Bioconductor Discovery [ (DAVID)24]. Genes (http://da- were classi- vid.abcc.ncifcrf.gov/fied using the Database (accessed for Annotation, on 21 November Visualization, 2018 and)) Integrated and Medline Discovery database (DAVID) (http://www.ncbi.nlm.nih.gov/(http://david.abcc.ncifcrf.gov/ (accessed (accessed on 21 on November 21 November 2018 2018)))) [25].and Medline database (http://www.ncbi.nlm.nih.gov/ (accessed on 21 November 2018)) [25]. 2.8. Statistical Analyses 2.8. Statistical Analyses All data were analyzed using GraphPad Prism v.5.0 (GraphPad Software, San Diego, All data were analyzed using GraphPad Prism v.5.0 (GraphPad Software, San Diego, CA, USA) and are expressed as the mean ± standard deviation. Statistical significance was CA, USA) and are expressed as the mean ± standard deviation. Statistical significance was evaluated using one-way and two-way analyses of variance. Statistical significance was evaluated using one-way and two-way analyses of variance. Statistical significance was set set at p < 0.05. at p < 0.05.

3. Results3. Results 3.1. 3.1.Measurement Measurement of Cell of CellViability Viability To evaluateTo evaluate the thecytotoxicity cytotoxicity of PHMG of PHMG and and CMIT, CMIT, HPAEpiCs HPAEpiCs were were treated treated with with dif- differ- ferentent concentrations concentrations of of PHMG PHMG and and CMIT CMIT for 24, 48, and 72 h, and cell viabilityviability waswas measured.measured.As As shown shown in in Figure Figure1, 1, although although there there was was a a slight slight change change inin cellcell viability in HPAEp- HPAEpiCs iCs treatedtreated with 2 2 μg/mLµg/mL of of PHMG PHMG and and CMIT, CMIT, HPAEpiCs HPAEpiCs treat treateded with with 3 μg/mL 3 µg/mL or more or more showedshowed significantly signiﬁcantly decreased decreased cell viability. cell viability. In particular, In particular, cell viability cell viability was relatively was relatively re- ducedreduced by PHMG by PHMG exposure exposure in a time in a-dependent time-dependent manner, manner, whereas whereas a significant a signiﬁcant decrease decrease in cell inviability cell viability was observed was observed after 3 μg/mL after 3 µCMITg/mL exposu CMITre, exposure, regardless regardless of the treatment of the treatment time. Hencetime., 2 μg/mL Hence, of 2 µ PHMGg/mL ofand PHMG CMIT and were CMIT chosen were for chosen further for experiments, further experiments, including including gene expressiongene expression analysis, analysis,to investigate to investigate the cellular the and cellular molecular and molecular responses. responses.

FigureFigure 1. Evaluation 1. Evaluation of the of cell the viabilities cell viabilities of human of human pulmonary pulmonary alveolar alveolar epithelial epithelial cells cells(HPAEpiCs) (HPAEpiCs) afterafter exposure exposure to polyhexamethylene to polyhexamethylene guanidine guanidine (PHMG) (PHMG) and andchloromethylisothiazolinone chloromethylisothiazolinone (CMIT). (CMIT). HPAEpiCsHPAEpiCs were were incubated incubated with with various various doses doses of (A of) (PHMGA) PHMG or (B or) CMIT (B) CMIT for 24, for 48, 24, and 48, and 72 h. 72 Cell h. Cell viabilityviability was wasmeasured measured in triplicate. in triplicate. Statistically Statistically significant signiﬁcant differences differences were were analyzed analyzed using using one- one- ^^^ wayway analysis analysis of variance of variance (ANOVA; (ANOVA; ** p < ** 0.01,p < *** 0.01, p < *** 0.001,p < 0.001,**** p < **** 0.0001p < v 0.0001ersus versus0 μg/mL 0 µ24g/mL h; p 24 h; < 0.001, ^^^^ p < 0.0001 versus 0 μg/mL 48 h; +++ p < 0.001, ++++ p < 0.0001 versus 0 μg/mL 72 h). ˆˆˆ p < 0.001, ˆˆˆˆ p < 0.0001 versus 0 µg/mL 48 h; +++ p < 0.001, ++++ p < 0.0001 versus 0 µg/mL 72 h).

3.2. 3.2.Comparative Comparative Analysis Analysis of Gene of Gene Expression Expression after after PHMG PHMG and andCMIT CMIT Exposure Exposure To date, no comparative study has analyzed the effects of PHMG and CMIT on gene expression. Thus, we performed RNA-Seq to analyze whether gene expression was altered by exposure to PHMG and CMIT. HPAEpiCs were incubated with 2 µg/mL PHMG and CMIT for the indicated time periods prior to gene expression analysis. Overall, the number of genes whose expression changed following exposure to PHMG and CMIT increased markedly over time, by circadian rhythm when exposed for 24 h (Figure S1A,B), and decreased by DNA replication and repair when exposed for 48 h (Figure S1C,D). Interestingly, genes that were signiﬁcantly upregulated and downregulated after PHMG Toxics 2021, 9, x FOR PEER REVIEW 5 of 11

To date, no comparative study has analyzed the effects of PHMG and CMIT on gene expression. Thus, we performed RNA-Seq to analyze whether gene expression was altered by exposure to PHMG and CMIT. HPAEpiCs were incubated with 2 μg/mL PHMG and CMIT for the indicated time periods prior to gene expression analysis. Overall, the number of genes whose expression changed following exposure to PHMG and CMIT increased markedly over time, by circadian rhythm when exposed for 24 h (Figure S1A,B), and decreased by DNA replication and repair when exposed for 48 h (Figure S1C,D). In- Toxics 2021, 9, 203 terestingly, genes that were significantly upregulated and downregulated5 of 10after PHMG exposure compared with those after CMIT exposure were selected (more than 3-fold; p < 0.05). In total, 34 genes were altered in HPAEpiCs treated with PHMG when compared with those in HPAEpiCs treated with CMIT; of these, a considerable proportion was re- exposure comparedlated to mineral with those absorption after CMITand mitochondrial exposure were ribosomal selected RNA (more (Figure than 2A). 3-fold; Notably, gene p < 0.05). Inontology total, 34 genesenrichment were altered analysis in HPAEpiCsshowed that treated genes with involved PHMG in when cellular compared responsiveness to with thoseZn in2+ HPAEpiCs were significantly treated withupregulated CMIT; ofin PHMG these, a-treated considerable HPAEpiCs proportion (Figure was2B). Among the related to mineralhuman absorptionMT1 genes and[26], mitochondrial our gene analysis ribosomal revealed RNA that (Figure the expression2A). Notably, levels of MT1B, gene ontologyMT1E, enrichment MT1F, MT1G analysis, MT1H showed, MT1M, that genes and MT1X involved were in cellularmarkedly responsiveness increased in HPAEpiCs 2+ to Zn weretreated signiﬁcantly with PHMG upregulated, but not in in PHMG-treated those treated with HPAEpiCs CMIT (Figure (Figure 2A).2B). Among the human MT1 genes [26], our gene analysis revealed that the expression levels of MT1B, MT1E, MT1F, MT1G, MT1H, MT1M, and MT1X were markedly increased in HPAEpiCs treated with PHMG, but not in those treated with CMIT (Figure2A).

Figure 2. Effects of PHMG and CMIT on the gene expression of HPAEpiCs. (A) Heatmap of selected genes (moreF thanigure 3-fold; 2. Effectsp < of 0.05) PHMG in HPAEpiCsand CMIT on after the exposure gene expression to 2 µg/mL of HPAEpiCs. of PHMG (A or) He CMIT.atmap of selected genes (more than 3-fold; p < 0.05) in HPAEpiCs after exposure to 2 µg/mL of PHMG or CMIT. (B) (B) Gene ontology enrichment analysis of the top 10 biological processes for selected genes. Gene ontology enrichment analysis of the top 10 biological processes for selected genes. 3.3. Upregulation of MT1B, MT1F, MT1G and MT1H in Cells Treated with PHMG but Not in Cells Treated3.3. with Upregulation CMIT of MT1B, MT1F, MT1G and MT1H in Cells Treated with PHMG but Not in To conﬁrmCells Treated the mRNA with expressionCMIT of different MT1 isomers identiﬁed in the gene expression analysis, HPAEpiCs were treated with PHMG or CMIT for the indicated time periods (12, 24, 48, and 72 h) and mRNA expression levels of MT1B, MT1E, MT1F, MT1G, MT1H, MT1M, and MT1X were measured using RT-PCR. As shown in Figure3, the mRNA

levels of MT1B, MT1F, MT1G, and MT1H were significantly increased in the cells treated with PHMG, but not in those treated with CMIT. No upregulation of MT1E, MT1M, or MT1X was detected in PHMG-treated and CMIT-treated cells. MT2 is a branch of MT1 and is composed of 61 amino acids. MT2 can be distinguished from MT3, which is composed of 68 amino acids and MT4, which is composed of 62 amino acids [27]. The expression of MT2 was also confirmed, but there was no significant difference between the PHMG- and CMIT-treated cells (Figure3). Collectively, these data indicate that PHMG, but not CMIT, specifically induces intracellular MT1B, MT1F, MT1G, and MT1H. Toxics 2021, 9, x FOR PEER REVIEW 6 of 11

To confirm the mRNA expression of different MT1 isomers identified in the gene expression analysis, HPAEpiCs were treated with PHMG or CMIT for the indicated time periods (12, 24, 48, and 72 h) and mRNA expression levels of MT1B, MT1E, MT1F, MT1G, MT1H, MT1M, and MT1X were measured using RT-PCR. As shown in Figure 3, the mRNA levels of MT1B, MT1F, MT1G, and MT1H were significantly increased in the cells treated with PHMG, but not in those treated with CMIT. No upregulation of MT1E, MT1M, or MT1X was detected in PHMG-treated and CMIT-treated cells. MT2 is a branch of MT1 and is composed of 61 amino acids. MT2 can be distinguished from MT3, which is composed of 68 amino acids and MT4, which is composed of 62 amino acids [27]. The expression of MT2 was also confirmed, but there was no significant difference between the PHMG- and CMIT-treated cells (Figure 3). Collectively, these data indicate that PHMG, but not CMIT, specifically induces intracellular MT1B, MT1F, MT1G, and MT1H.

Toxics 2021, 9, 203 6 of 10

Figure 3. Effects of PHMG and CMIT on the mRNA expression of metallothionein-1 (MT1). (A) At various time points after exposure to 2 µg/mL of PHMG and CMIT, the mRNA expression levels of MT1B, MT1E, MT1F, MT1G, MT1H, MT1M, MT1X, and MT2A were analyzed through reverse transcription-polymerase chain reaction (RT-PCR). (B) Quantitative analysis of mRNA expression. Statistically signiﬁcant differences were analyzed by one-way analysis of variance (ANOVA; * p < 0.05 and *** p < 0.001). Figure 3. Effects of PHMG and CMIT on the mRNA expression of metallothionein-1 (MT1). (A) At 3.4.various Role time of MTF1 points in after the exposure Induction to of 2 MT1B,µg/mL of MT1F, PHMG MT1G, and CMIT, and MT1Hthe mRNA expression levels of MT1B,Previous MT1E, MT1F, studies MT1G, have MT1H, shown MT1M, that MTF1MT1X, playsand MT2A a pivotal were analyzed role in the through induction reverse of tran- MTs, whichscription regulate-polymerase intracellular chain reaction metal (RT homeostasis-PCR). (B) Quantitative [13]. In this analysis study, weof mRNA observed expression. the nuclear Sta- translocationtistically significant of MTF1 differences protein were by analyzed Western by blotting one-way afteranalysis exposure of variance to PHMG(ANOVA and; * p < CMIT 0.05 inand HPAEpiCs. *** p < 0.001). In a time-course study, the expression level of translocated MTF1 peaked after 4 h in PHMG-treated HPAEpiCs (Figure4). In contrast, no nuclear translocation of 3.4. Role of MTF1 in the Induction of MT1B, MT1F, MT1G, and MT1H MTF1 was observed in the CMIT-treated HPAEpiCs, suggesting that the differences in the expressionPrevious of MT1B,studies MT1F, have shown MT1G, that and MTF1 MT1H plays after a PHMGpivotal androle in CMIT the induction exposure of resulted MTs, fromwhich differences regulate intracellular in transcriptional metal homeostasis regulation of [13] MTF1.. In this study, we observed the nuclear translocationThe MT1 of promoters MTF1 protein contain, by W bindingestern blotting sites for after Zn-dependent exposure to PHMG transcription and CMIT factors in (ZTFs),HPAEpiCs. such In as a MTF1,time-course EGR1, study, and the NR3C1, expression which level have of Zntranslocated2+-binding MTF1 motifs peake [27].d after Most MT1 isomers have binding sites for both EGR1 and NR3C1, but MT1F only has an EGR1 binding site and MT1B and MT1H only have NR3C1 binding sites [27]. MT1G also has no binding sites for EGR1 or NR3C1 [27]. Therefore, we hypothesized that the activation of

MTF1, which regulates specific MT1 isomer expression, requires the transcriptional activity of EGR1 and NR3C1. To address this hypothesis, we investigated the protein expression levels of EGR1 and NR3C1 after treatment of HPAEpiCs with PHMG and CMIT. Herein, we observed that the nuclear translocation of EGR1 was significantly increased at 1 h and 2 h in HPAEpiCs treated with PHMG and CMIT, respectively. (Figure4). In addition, the nuclear translocation of NR3C1 was also significantly upregulated after PHMG treatment in a time- dependent manner, whereas the nuclear translocation of NR3C1 was slightly increased at 4 h in CMIT-treated HPAEpiCs (Figure4). Thus, differences in the expression of MT1B, MT1F, and MT1H after PHMG and CMIT exposure were not significantly affected by EGR1 and NR3C1 transcriptional regulation after PHMG and CMIT exposure, suggesting that EGR1 and NR3C1 may be the basic transcription factors for the upregulation of MT1B, MT1F, and MT1H. Because MT1G has no binding sites for EGR1 or NR3C1 [27], the expression of MT1G could be regulated by MTF1. Taken together, these results indicate that MTF1 may be an essential transcription factor for the induction of MT1B, MT1F, MT1G, and MT1H after exposure to PHMG. Toxics 2021, 9, x FOR PEER REVIEW 7 of 11

4 h in PHMG-treated HPAEpiCs (Figure 4). In contrast, no nuclear translocation of MTF1 was observed in the CMIT-treated HPAEpiCs, suggesting that the differences in the expression of MT1B, MT1F, MT1G, and MT1H after PHMG and CMIT exposure resulted Toxics 2021, 9, 203 from differences in transcriptional regulation of MTF1. 7 of 10

Figure 4. Effects of PHMG and CMIT on the expression levels of MTF1, EGR1, and NR3C1. HPAEpiCs were treated with 2 µ g/mL of PHMG or CMIT for the indicated time points. (A) Protein levels of metal regulatory transcription factor 1 (MTF1); early growth response 1 (EGR1); nuclear receptor superfamily 3, group C, member 1 (NR3C1); Lamin B; and α-tubulin were Figure 4. Effects of PHMG and CMIT on the expression levels of MTF1, EGR1, and NR3C1. HPAEp- measured using Western blotting at 0, 0.5, 1, 2 and 4 h after exposure to PHMG and CMIT. Lamin B and α-tubulin were iCs were treated with 2 µg/mL of PHMG or CMIT for the indicated time points. (A) Protein levels used as nuclear and cytosol loadingof metal controls, regulatory respectively. transcription (B) Quantitativefactor 1 (MTF1) analysis; early ofgrowth the protein response levels 1 (EGR1) of MTF1,; nuclear EGR1, receptor and NR3C1. Statistically significantsuperfamily differences 3, group were C, analyzed member by 1 one-way(NR3C1) ANOVA; Lamin B(*; andp < 0.05,α-tubulin** p < were 0.01, measured and *** p < using 0.001). Western blotting at 0, 0.5, 1, 2 and 4 h after exposure to PHMG and CMIT. Lamin B and α-tubulin were used 4.as Discussion nuclear and cytosol loading controls, respectively. (B) Quantitative analysis of the protein levels of MTF1,In human EGR1, and and animalNR3C1. Statistically studies, the significant inhalation differences of PHMG were is analyzed strongly by associated one-way ANOVA with lung(* p injuries,< 0.05, ** includingp < 0.01, and interstitial *** p < 0.001 pneumonitis). and lung fibrosis, which eventually lead to death owing to the loss of lung function [1,28–30]. Lung injuries occur in people exposed to PHMG,The aMT1 representative promoters HDcontain substance;, binding however, sites for lessZn- severedependent lung transcription damage has beenfactors reported(ZTFs), such in people as MTF1, exposed EGR1, to and CMIT NR3C1, [4]. which Nonetheless, have Zn the2+-binding differences motifs in [27] the. Most toxicity MT1 mechanismsisomers have of chemicalsbinding sites used for in both HDs EGR1 and their and rolesNR3C1, in the but development MT1F only has of lungan EGR1 diseases bind- areing poorly site and understood. MT1B and In MT1H this study, only have we analyzed NR3C1 binding the differential sites [27] gene. MT1G expression also has of no HPAEpiCsbinding sites after for treatment EGR1 or with NR3C1 PHMG [27] and. Therefore, CMIT and we elucidatedhypothesized the that differences the activ ination their of molecularMTF1, which responses regulates to these specific agents. MT1 isomer expression, requires the transcriptional activity Theof EGR1 results and of theNR3C1. analysis To ofaddress gene expression this hypothesis, in human we alveolarinvestigated A549 the cells protein after PHMG expres- exposuresion levels have of already EGR1 and been NR3C1 reported after [10 ].treatment However, of the HPAEpiCs previous resultswith PHMG are significantly and CMIT. differentHerein, fromwe observed our genetic that analysisthe nuclear results translocation because of of theEGR1 different was significantly cell type and increased PHMG at dose1 h usedand 2 forh in genetic HPAEpiCs analysis. treated To date,with PHMG CMIT-induced and CMIT, gene respectively. expression (Figure has not 4). yet In been addi- studied.tion, the Therefore, nuclear translocation we performed of gene NR3C1 expression was also analysis significantly after treating upregulated HPAEpiCs after PHMG with PHMGtreatment and CMIT.in a time Unlike-dependent CMIT-induced manner, genewhereas sets, the most nuclear PHMG-induced translocation gene of NR3C1 sets were was associated with cellular responses to metal ions, especially Zn2+. Zn2+ is an essential ion that activates multiple intracellular enzymes and regulates the activity of transcription factors. In particular, methallothionein1 isomers including MT1B, MT1E, MT1H, MT1HL1, and MT1X, and the membrane transporter gene SLC30A1, which controls the concentration of intracellular Zn2+ [26], were upregulated in cells exposed to PHMG, while PARP1, which is involved in DNA repair [31], was downregulated in HPAEpiCs exposed to PHMG and Toxics 2021, 9, 203 8 of 10

CMIT (more than 2-fold; p < 0.05). Notably, our gene expression analysis data demonstrated that PHMG—but not CMIT—significantly induced the expression of MT1, which is capable of binding to metal ions and inhibiting the cellular stress caused by dysregulation of intracellular metal ion concentration [13]. Our results revealed that the mRNA expression of MT1B, MT1F, MT1G, and MT1H was markedly upregulated in the HPAEpiCs treated with PHMG but not in those treated with CMIT, suggesting that the suppression of MT1B, MT1F, MT1G, and MT1H is responsible for the discrepancy in the cellular responses between the PHMG and CMIT groups. Thus, our results indicate that, compared with CMIT, PHMG significantly increased the expression levels of specific MT1 factors, namely MT1B, MT1F, MT1G, and MT1H. Several studies have reported that MT1 treatment alleviates lung injury caused by inhaled toxins, such as lipopolysaccharide, Ni, ozone, and paraquat [15–17,32]. Obviously, the increase of oxidative stress is detected in the lung tissue of MT-I/II null mice compared to wild-type mice [15,16,18,33], suggesting that MT1 has essential roles in the protection of cells under oxidative stress. Under cellular oxidative stress, MT1 binds with reactive oxygen species and simultaneously releases Zn2+ into the cytosol, and the consequent facilitation of the binding of MTF1 and Zn2+ [34]. These results suggest that an increase in the expression level of MT1 in cells owing to exposure to PHMG is beneficial for cell survival. In contrast, some lung cancer types, such as large cell lung cancer and non-small cell lung cancer, exhibit MT1 upregulation [19]. In addition, the overexpression of MT1 and MT2 has been detected in tumor tissue samples of lung carcinomas, including squamous cell lung carcinoma and adenocarcinoma [20]. Notably, our recent study showed that bronchiolar-alveolar adenomas are increased in rats after PHMG intratracheal injection [35]. Hence, the upregulation of MT1B, MT1F, MT1G, and MT1H in HPAEpiCs after exposure to PHMG may be involved in the development of lung cancer. Our results showed that alterations in the expression levels of epithelial mesenchymal transition-related genes associated with cancer progression and metastasis were not significantly different between cells treated with PHMG and CMIT. However, mitochondrial large subunit ribosomal RNA (MTRNR2) was shown to be as much upregulated in cells exposed to PHMG as MT1 (Figure2). MTRNR2 is a 16S ribosomal RNA in mitochondria that encodes a small open reading frame called humanin (HN), which binds to pro-apoptotic proteins such as Bax, tBid, and BimEL to suppress the induction of cytochrome c, thus inhibiting apoptosis [36,37]. A recent study reported that the additional treatment of HN in mice with breast cancer cells increases spontaneous lung metastasis [38]. In addition, MT1 inhibits apoptosis and promotes cancer cell survival [34]. Therefore, MT1 and MTRNR2 interactions may play an important role in lung cancer development. There are multiple cis-acting elements for ZTFs, such as MTF1, EGR1, NR3C1, speci- ficity protein 1 (SP1), retinoic acid receptor (RAR), Ikaros family zinc finger protein 1 (IKZF1), and churchill domain containing 1 (CHURC1) in the promoter region of MT1 [27]. It is well known that MTF1 plays an important role in the induction of MTs [27]. Among the MT1 isomers, MT1B and MT1H only have a binding site for NR3C1, whereas MF1F only has a binding site for EGR1. However, MT1G has no binding sites for EGR1 or NR3C1 [27]. Therefore, the possibility of regulating the expression of MT1F via EGR1 and MT1B and MT1H via NR3C1 could not be excluded; thus, the protein levels of EGR1 and NR3C1 were investigated. Our results showed that the expression levels of EGR1 and NR3C1 were increased in the HPAEpiCs treated with PHMG and CMIT, indicating that EGR1 and NR3C1 play a pivotal role in upregulating MT1B, MT1F, and MT1H. In particular, MT1G, which does not have a binding site for EGR1 and NR3C1 [27], could be affected by the transcriptional regulation of MTF1. Thus, our results suggest that the upregulation of MT1B, MT1F, MT1G, and MT1H results from the exposure of cells to PHMG and is regulated by MTF1. However, EGR1 and NR3C1 are not prerequisites for the induction of MT1G upon exposure to PHMG. Although EGR1 and NR3C1 are induced by CMIT exposure, it cannot lead to the upregulation of MTF1, suggesting that MTF1 is a vital transcription factor, along with EGR1 and NR3C1, for the expression of MT1B, MT1F, MT1G, and MT1H. Toxics 2021, 9, 203 9 of 10

5. Conclusions Compared to CMIT, PHMG causes two prime cellular responses. First, it induces the synthesis of speciﬁc MT1 isomers, including MT1B, MT1F, MT1G, and MT1H. Second, MTF1 is an essential transcription factor that induces the expression of MT1B, MT1F, MT1G, and MT1H. To our knowledge, this is the ﬁrst study to report the differences in the cellular responses of PHMG and CMIT. The data suggest that the differences in the molecular mechanisms activated by PHMG and CMIT can be used to explain the differences in lung injuries that occur when these agents are inhaled. Therefore, it is necessary to investigate the expression of MT1 isomers and their transcriptional regulation mechanisms after exposure to PHMG and CMIT in future animal studies.

Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/toxics9090203/s1, Figure S1: Effects of PHMG and CMIT on the gene expression of HPAEpiCs. Author Contributions: Conceptualization, S.-H.J. and J.K.; Methodology, S.-H.J. and Y.-J.N.; Vali- dation, J.K., C.K., H.L. (Hong Lee), Y.-W.B. and E.-K.P.; Investigation, S.-H.J., Y.-J.N., A.T., J.-Y.K., J.-Y.C., H.L. (Hyejin Lee) and M.-O.S.; Writing–Original Draft Preparation, S.-H.J. and J.-H.L.; Writing– Review & Editing, J.K., C.K. and K.-Y.L.; Supervision, J.-H.L. and K.-Y.L.; Project Administration, J.-H.L.; Funding Acquisition, J.-H.L. and K.-Y.L. All authors contributed to this study. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by a grant from the National Institute of Environment Research (NIER) funded by the Ministry of Environment (MOE) of the Republic of Korea (grant number NIER-2021-04-03-001) and the Korea University Grant (grant number K1813161). Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Data is contained within article. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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